Rina Perlman

The effect of glucose 2-deoxy-D-glucose and insulin on estradiol secretion by cultured human trophoblast

Abstract Human trophoblasts were isolated in a monolayer cell culture and the effect of extra-and intracellular glucopenia on estradiol secretion was studied. Term placentas were dispersed by repeated short term trypsinization and 2×10 6 cells plated in each dish. The de novo synthesis of estradiol was demonstrated by a 10 fold increase in estradiol secretion by supplementation of androstendione. Incubation with dibutyryl cyclic AMP produced a dose dependent increase in estradiol secretion. Low glucose (10 mg/dl) medium enhanced estradiol secretion when compared to a medium containing 50–100 mg/dl glucose. Intracellular glucopenia by 2-deoxyglucose produced an increase in estradiol secretion. The results indicate negative dependency of estradiol secretion by the trophoblast on intracellular glucose.

The dual effect of calcium on aromatization by cultured human trophoblast

To study the effect of calcium ion on aromatization of an androgenic precursor to estradiol by the placenta, cultured term trophoblasts were used as a model system. Secretion of estradiol into the culture medium was regarded as indicating aromatization, since cells cultured with no androgenic precursors produced only insignificant amounts of estradiol. EGTA, verapamil and ionophore A23187 inhibited aromatization, while trifluoperazine, an inhibitor of the calcium-calmodulin complex, interfered with the stimulatory effect of cyclic AMP on aromatization. We conclude that calcium ion has an essential role in the aromatization of 4-androstene-3,17-dione to estradiol. The calcium-calmodulin complex is required for activation of aromatase by cyclic AMP. However, when flooded with calcium by ionophore A23187, the trophoblast is unable to effectively buffer calcium, and aromatization is inhibited.

Two pathways of placental lactogen secretion by cultured human trophoblast

To assess the relative importance of regulated and of constitutive secretion of placental lactogen, a cell culture model of term human trophoblast was utilized. Time courses of secretion revealed a constant secretion rate over 9 days of culture, with relatively small constant intracellular hormone concentration. Potassium, 21 mM, produced a slight but significant increase in hormone secretion into the medium. Growth hormone-releasing hormone (5 X 10(-10)-5 X 10(-9)) stimulated a 27-48% increase in placental lactogen secretion. The data suggest a major process of constitutive secretion and a minor role for regulated secretion from a storage pool.

Effects of human growth hormone upon term placental hormone secretion in vitro.

The role of human growth hormone (hGH) on placental hormone secretion at term was investigated in two in vitro models: placental explants and cultured trophoblastic cells. Physiological concentrations of hGH caused a significant dose-dependent increase in placental lactogen and progesterone secretion. In the explant model it stimulated estradiol secretion. In order to determine whether this stimulatory effect on estradiol is exerted via aromatase, an isolated cell culture was utilized where androstenedione was supplied as substrate. In this model, hGH exerted a mild inhibitory effect. In conclusion, hGH at levels present in the fetal circulation exerts a significant stimulatory effect upon placental function as reflected by both peptide and steroid hormone production and secretion. The effect of estradiol secretion is the end result of an inhibitory effect on androgen aromatization and a stimulatory effect on earlier steps.

The effect of inhibitors on insulin regulation of hormone secretion by cultured human trophoblast

The effect of insulin in physiological concentrations on hormone secretion by human term trophoblast cell culture was studied in relation to insulin control of glucose transport and utilization. Specific inhibitors to these 2 functions were added to the culture, either alone to test for a dose response or with insulin. 2-Deoxy-D-glucose, which inhibits glycolysis, did not change the pattern of response of estradiol and placental lactogen (hPL) secretion to insulin. Phloridzin, an inhibitor of glucose transport, interfered with the stimulatory effect of insulin on hPL secretion, but stimulated insulin inhibition of estradiol secretion when added by itself. The results indicate that hPL response to insulin is dependent on glucose transport, but is independent of glycolysis. Estradiol response is dependent on glucose transport, but is independent of glycolysis.

The modulation of placental lactogen secretion by calcium: studies with cultured human term trophoblast

Previous reports have documented the negative regulatory effect of calcium ion on the secretion of human placental lactogen by the placenta. Human term trophoblasts were dispersed from term placenta and maintained in monolayer cell culture. Incubation of the cultured trophoblast with EGTA produced a dose-dependent stimulation of placental lactogen secretion. The calcium ionophore A23187 inhibited hormone secretion. This inhibitory effect was eliminated by the addition of the calcium-calmodulin complex inhibitor trifluoperazine. It is concluded that calcium exerts a negative regulatory effect on the secretion of placental lactogen by a mechanism which requires the action of the calcium-calmodulin complex.

The human placenta as a target tissue for glucagon

Glucagons effect on the placenta was studied in cultured human term trophoblast and in homogenized term and first-trimester placentas. In studies with cultured term trophoblast, glucagon stimulated the generation of cyclic AMP and estradiol secretion and inhibited placental lactogen secretion. Incubation of homogenates of term and of first-trimester placenta with 0.5 mM dibutyryl cAMP revealed a marked decrease of pyruvate kinase activity. Glucagon produced a similar decrease in first-trimester homogenates, but failed to affect term placentas. The present demonstration of the placenta as a target tissue for glucagon suggests an active contribution of the trophoblast to energy metabolism during pregnancy.